Rewiring Photosynthesis by Water‐Soluble Fullerene Derivatives for Solar‐Powered Electricity Generation

Abstract Natural photosynthesis holds great potential to generate clean electricity from solar energy. In order to utilize this process for power generation, it is necessary to rewire photosynthetic electron transport chains (PETCs) of living photosynthetic organisms to redirect more electron flux toward an extracellular electrode. In this study, a semi‐artificial rewiring strategy, which use a water‐soluble fullerene derivative to capture electrons from PETCs and donate them for electrical current generation, is proposed. A positively charged fullerene derivative, functionalized with N,N‐dimethyl pyrrolidinium iodide, is found to be efficiently taken up by the cyanobacterium Synechocystis sp. PCC 6803. The distribution of this fullerene derivative near the thylakoid membrane, as well as site‐specific inhibitor assays and transient absorption spectroscopy, suggest that it can directly interact with the redox centers in the PETCs, particularly the acceptor side of photosystem I (PSI). The internalized fullerene derivatives facilitate the extraction of photosynthetic electrons and significantly enhance the photocurrent density of Synechocystis by approximately tenfold. This work opens up new possibility for the application of fullerenes as an excellent 3D electron carrier in living biophotovoltaics.

The fitted kinetic parameters were summarized in Table S2.

Figure S2 .
Figure S2.The absorption spectra of Synechocystis sp.PCC 6803 and C60-DMePyI aqueous solutions.The concentration proportions of Synechocystis and C60-DMePyI corresponds to those in incubation mixtures used for photocurrent measurement.

Figure S3 .
Figure S3.Stepped chronoamperometry scans on Synechocystis biofilms under chopped light irradiation.The photoresponses profiles of bare electrode (ITO glass), Synechocystis alone and Synechocystis treated with C60-DMePyI were recorded under different applied potentials of working electrode.The gray columns indicate light off.A monochromatic red light source (λ = 658 nm) with an intensity of 400 μmol photons m -2 s -1 was used.

Figure S4 .
Figure S4.The photocurrents generated by Synechocystis cells alone under different light intensities.

Figure S6 .
Figure S6.Long-term performance of photocurrent generation.a) Photocurrent generation over thirty light/dark cycles (~2 h).b) Successive photocurrent generation over thirty light/dark cycles after kept the electrochemical devices at room temperature for 12 h.

Figure S7 .
Figure S7.Cytotoxicity assays of C60-DMePyI.a) Cytotoxicity spot assay.Synechocystis cells were incubated with C60-DMePyI at different concentrations for 12 h.Serial dilutions were prepared, spotted on BG11 agar plate and cultured for one week.b) Fluorescence intensity of PI stained Synechocystis cells that treated with C60-DMePyI at different concentrations.The dead cells that treated with 70% isopropanol for half an hour was used as positive control.The excitation and emission wavelengths were set at 485 nm and 630 nm, respectively.The data are presented as mean values ± SD from independent biological replicates (n = 3).Statistical significance was analyzed using unpaired two-tailed Student's t-test, and significance levels were denoted as *p <0.05, **p <0.01, ***p <0.001.

Figure S8 .
Figure S8.The effects of site-specific inhibitors on photocurrent generation of Synechocystis alone.a) The photocurrent generation under DCMU inhibition.b) The photocurrent generation under DBMIB inhibition.c) The photocurrent generation under PMA inhibition.d) The photocurrent generation under HQNO inhibition.

Figure S9 .
Figure S9.Ferredoxin redox assays.a) SDS-PAGE of purified Synechocystis ferredoxin heterologously expressed in E. coli BL21(DE3).b) In vitro re-oxidation of dithionite-reduced ferredoxin by C60-DMePyI under anaerobic conditions.The UV-visible absorption spectra of ferredoxin at different states were recorded.The oxidized ferredoxin shows the characteristic absorption peaks at near 330 nm, 420 nm and 460 nm.

Figure S10 .
Figure S10.The effects of C60-DMePyI on the photochemical quantum efficiencies of PSII and PSI.a) The complementary quantum yields of PSII, including Y(II), Y(NO) and Y(NPQ).Y(II) represents the effective photochemical quantum yield of PSII, Y(NO) represents the quantum yield of non-light-induced non-photochemical fluorescence quenching and Y(NPQ) represents the quantum yield of light-induced non-photochemical fluorescence quenching.b) The complementary quantum yields of PSI, including Y(I), Y(ND) and Y(NA).Y(I) represents the effective photochemical quantum yield of PSI, Y(ND) represents the quantum yield of non-photochemical energy dissipation due to donor side limitation and Y(NA) represents the quantum yield of non-photochemical energy dissipation due to acceptor side limitation.The data are presented as mean values ± SD from independent biological replicates (n = 3).

Figure S11 .
Figure S11.The OJIP fluorescence transients of Synechocystis alone and the Synechocystis treated with C60-DMePyI.The data are presented as mean values ± SD from independent biological replicates (n = 10).

Figure S12 .
Figure S12.Fluorescence emission spectra of Synechocystis recorded under open and closed PSII reaction centers.Minimal fluorescence emission (F 0 ) under open reaction centers and maximal fluorescence emission (F M ) under closed reaction centers were recorded at the excitation wavelength of 435 nm (a), 475 nm (c) and 570 nm (e).Variable fluorescence (F V = F M -F 0 ) for excitation at 435 nm (b), 475 nm (d) and 570 nm (f) were calculated accordingly.The spectra were normalized between 0 and 1.The concentration of C60-DMePyI was 0.1 mg mL -1 .

Figure S14 .
Figure S14.Transient absorption kinetics of Synechocystis at 690 nm.a) Transient absorption kinetics at time scale of -2 to 100 ps.b) Transient absorption kinetics at time scale of -2 to 20 ps.Square scatters represent original data and solid lines indicate fit curves (bi-exponential).

Figure S17 .
Figure S17.The photographs of aqueous solutions of different fullerene derivatives before and after incubation with Synechocystis cells.

Figure S18 .
Figure S18.Fluorescence emission spectra of Synechocystis after incubated with different fullerene derivatives.a) Minimal fluorescence emission (F 0 ) recorded under open PSII reaction centers.b) Maximal fluorescence emission (F M ) recorded under closed PSII reaction centers.c) Variable fluorescence (F V = F M -F 0 ).d) Maximum quantum yield (F V /F M ).The excitation wavelength was set at 570 nm and the spectra were normalized between 0 and 1.The concentration of fullerene derivatives was 0.1 mg mL -1 .

Figure S19 .
Figure S19.Fluorescence emission spectra of Synechocystis treated with high-dose fullerene derivatives.The Synechocystis cells were treated with different fullerene derivatives at a concentration of 1.0 mg mL -1 for half an hour.The residual fullerene derivatives in the supernatants were washed out before measurements.The fluorescence emission was recorded under open PSII reaction centers (F 0 ) at an excitation wavelength of 570 nm.

Figure S20 .
Figure S20.The absorption spectra of Synechocystis cells after incubated with different fullerene derivatives at a concentration of 1.0 mg mL -1 .

Table S1 .
Summary of the JIP-test for OJIP fluorescence transients.

Table S2 .
The fitted kinetic parameters of TA spectroscopy of Synechocystis.